Aberrant cytoplasmic sequestration has been reported as an alternative mechanism of p53 inactivation to mutation in neuroblastoma. We hypothesized that p53 localization and function in neuroblastoma ...is related to differentiation status. Eighty-two untreated and 24 paired pre and post-chemotherapy neuroblastomas were studied by immunocytochemistry for p53, p21
WAF1
, BAX, Bcl2 and Ki67. Predominantly nuclear p53 was detected in undifferentiated neuroblastoma, and both nuclear and cytoplasmic p53 in differentiating neuroblastoma. The nuclear p53 labeling index (LI) correlated with the Ki67 LI (r = 0.51, p
p53 mutations are rare in neuroblastomas at diagnosis perhaps accounting for their initial response to therapy, but advanced neuroblastoma frequently relapses, and it is possible that p53 mutations ...develop later. Two neuroblastoma cell lines derived from the same patient before SKNBE(1n) and after SKNBE(2c) cytotoxic therapy were analyzed for the presence of chromosome 17 and p53 genes by fluorescent in situ hybridization, p53 mutations by DNA sequencing, and p53 function after irradiation by studying the transcription of p53-regulated genes, cell cycle arrest, and induction of apoptosis. The SKNBE(1n) cell line was wild-type for p53, had two p53 genes, two copies of chromosome arm 17p and showed functional p53 after irradiation. The SKNBE(2c) cell line derived from the same patient 5 months later at relapse had loss of an entire chromosome 17, resulting in hemizygosity for the p53 locus on 17p and a missense p53 mutation in exon 5, and p53 was not functional after irradiation. The appearance of a p53 mutation in a cell line derived from a relapsed neuroblastoma suggests that this may be a mechanism of resistance to therapy. If p53 mutations develop frequently in relapsed neuroblastoma, cytotoxic agents more sensitive to mutant p53 might be more effective at relapse.
Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize ...fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-β/γ-specific antagonists, but not by an RARα-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.
Studies involving small case series have suggested that malignant fibrous histiocytoma of bone (MFH-B) is a chemosensitive tumor and that chemotherapy may improve survival. In this study, we ...evaluated clinical and pathologic response rates and survival in a series of patients treated with a consistent chemotherapy regimen of doxorubicin and cisplatin (DOX/DDP).
Study patients were required to have biopsy-proven MFH-B, no previous chemotherapy, and primary or metastatic measurable disease and to be </= 65 years of age. Treatment consisted of doxorubicin 25 mg/m(2)/d days 1 through 3 and cisplatin 100 mg/m(2) by 4-hour intravenous infusion every 3 weeks for six cycles. In patients with operable primary tumors, chemotherapy was planned to start within 42 days of biopsy, with definitive surgery performed after three cycles.
Forty-one patients had operable nonmetastatic limb sarcomas, and 23 (56%) completed six chemotherapy cycles. Limb salvage was possible in 33 patients (80%), and 16 (42%) of 38 assessable specimens showed a good pathologic response (>/= 90% necrosis). Median time to progression was 56 months, and the 5-year progression-free survival rate was 56% (95% confidence interval CI, 40% to 72%). Median survival time was 63 months, and the 5-year survival rate was 59% (95% CI, 41% to 77%). Patients with a good pathologic response had longer survival times and times to progression than did those with a poor response. Also treated were two patients with locally recurrent and nine with metastatic disease, and these patients had a median survival time of 17.5 months.
Our study suggests that adjuvant or neoadjuvant chemotherapy with DOX/DDP is beneficial in MFH-B. Good pathologic response rates and survivals are quite comparable with those for osteosarcoma, a related bone tumor for which adjuvant or neoadjuvant chemotherapy is an accepted practice.
This study investigated the hypothesis that p53 accumulation in neuroblastoma, in the absence of mutation, is associated with functional inactivation, which interferes with downstream mediators of ...p53 function. To test this hypothesis, p53 expression, location, and functional integrity was examined in neuroblastoma by irradiating 6 neuroblastoma cell lines and studying the effects on p53 transcriptional function, cell cycle arrest, and induction of apoptosis, together with the transcriptional function of p53 after irradiation in three ex vivo primary, untreated neuroblastoma tumors. p53 sequencing showed five neuroblastoma cell lines, two of which were MYCN-amplified, and that all of the tumors were wild-type for p53. p53 was found to be predominantly nuclear before and after irradiation and to up-regulate the p53 responsive genes WAF1 and MDM2 in wild-type p53 cell lines and a poorly-differentiated neuroblastoma, but not a differentiating neuroblastoma or the ganglioneuroblastoma part of a nodular ganglioneuroblastoma in short term culture. This suggests intact p53 transcriptional activity in proliferating neuroblastoma. Irradiation of wild-type p53 neuroblastoma cell lines led to G(1) cell cycle arrest in cell lines without MYCN amplification, but not in those with MYCN amplification, despite induction of WAF1. This suggests MYCN amplification may alter downstream mediators of p53 function in neuroblastoma.
A randomized, double‐blind, 1‐year pilot study of prednisolone treatment for primary biliary cirrhosis was undertaken. Nineteen patients received 30 mg prednisolone per day initially, with a ...maintenance dose of 10 mg per day. Seventeen patients received placebo. The groups were matched for age, menopausal status, hepatic histological stage and bilirubin.
Treatment was well tolerated without dropouts. Two patients receiving prednisolone developed diabetes, one a duodenal ulcer and one depression. One patient receiving placebo died of liver failure after 3 months.
Cholestatic symptoms (itch and fatigue) improved on prednisolone. There was significant (prednisolone vs. placebo) improvement in transaminase (p = 0.0214), alkaline phosphatase (p = 0.0032), procollagen III peptide (p = 0.0103), immunoglobulin G (p = 0.0012) and liver histology (p = 0.016); these changes were greatest among noncirrhotic patients.
No patient developed skeletal symptoms. Fifty‐seven per cent had abnormal triolein breath tests prior to treatment, and 65% had abnormally low calcium absorption tests. Calcium absorption increased significantly in the treated group vs. placebo at 2 weeks (p < 0.02), but not at 1 year. Femoral photon absorptiometry fell in the prednisolone group after 1 year (‐3.5% vs. placebo +0.5%, p < 0.05), as did trabecular bone volume (‐6% vs. ‐2.8%, p < 0.005) and resorption surface (‐11% vs. +2%, p < 0.02) on serial bone biopsy.
Prednisolone seems to exert a favorable hepatic effect in primary biliary cirrhosis but at the expense of increased bone loss to approximately twice the expected rate. Prednisolone treatment merits further assessment in primary biliary cirrhosis over a longer period, with attention to selection of patients most likely to benefit and continuing observation of bone mass to better establish the “cost/benefit” ratio.
Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-
trans and 9-
cis isomers having different biological properties. ...Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-
cis and all-
trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1α, TIF1β, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1α, TIF1β and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-
trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-
cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.
To date, the clinical success of 13-cis or all-trans retinoic acid in the treatment of neuroblastoma has been disappointing. In vivo, 13-cis will isomerise to both all-trans and 9-cis retinoic acid, ...believed to be the main biologically-active isomers. In vitro studies with an N-type neuroblastoma cell line, SH SY 5Y, show that 9-cis is better than other isomers at both inducing morphological differentiation and inhibiting proliferation. RAR-beta, a gene which may mediate retinoic acid responsiveness and be of prognostic significance, is also more-effectively induced by 9-cis retinoic acid. 9-cis and all-trans retinoic acid do not have synergistic effects on SH SY 5Y cell proliferation and gene expression. A retinoid X receptor (RXR)-specific analogue of 9-cis retinoic acid had similar effects on gene expression to 9-cis retinoic acid alone. In view of these results, 9-cis retinoic acid or stable analogues of this retinoid may have potential for the treatment of neuroblastoma.
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